Coupling of polyethylene glycol (PEG) to biologically active molecules termed “Pegylation” are used in the delivery biologically active molecules usually proteins and small molecules. At least one advantage of this process is modifying the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the biologically active molecules and improving therapeutic effectiveness of the biologically active molecules. Pegylation increases the size and molecular weight of proteins and small molecules resulting in the extension of their half-life in plasma. In general, Pegylation may alter the physicochemical properties of the proteins and therapeutic molecules resulting in decreased bioactivity of the parent proteins and organic therapeutic molecules. It is therefore desirable to optimize the PK and PD properties of PEG-protein conjugates for achieving their maximum therapeutic efficacies.
Covalently attaching PEG to biologically active molecules in the prior art, including linear and branched PEG polymers, has been achieved. In vast majority of cases amino groups of the biologically active molecules are utilized as the sites of attachment. US Patent Application 20030190304 to Thompson et al. describes Pegylation reagents. U.S. Pat. No. 7,030,278 to Harris et al. describes certain PEG derivatives with proximal reactive groups. Certain non-antigenic branched polymer conjugates are described in U.S. Pat. No. 5,643,575 to Martinez et al. Certain multi-armed PEG polymers are described in the US Patent Application 20050033058 to Huang et al. Certain active carbonates for modification of polypeptides are disclosed in U.S. Pat. No. 5,122,614 to Zalipsky. Interferon polymer conjugates utilizing stable (permanent) linear PEG polymers for conjugation are described in U.S. Pat. No. 5,711,944 to Gilbert et al. Interferon polymer conjugates utilizing stable branched PEG polymers for conjugation are described in U.S. Pat. No. 5,932,462 to Milton et al.
Protein drugs of molecular mass lower than 50,000 Daltons are in general short-lived species in vivo, having a circulatory half-life of about 5-20 min. Clearance of proteins occurs through several mechanisms, including glomerular infiltration in the kidney, receptor-mediated endocytosis, and degradation by peripheral tissues, and proteolysis at the tissue surfaces or by serum proteases. Considering also that protein drugs are not absorbed orally, prolonged maintenance of therapeutically active drugs in circulation is a desirable feature of primary clinical importance. This condition, however, is rarely achieved after a single administration of low molecular weight peptides and protein drugs. At least one strategy to achieve such goals described in the art is pegylating such biomolecules. However, the drawback in prior art Pegylation methodologies is the loss of biological activity of the proteins that are pegylated by conventional permanent branched or linear PEG compounds. Such loss of biological activity is due to the steric hindrance created by the large PEG polymers that are being attached to the biologically active protein of interest. For example, the pegylated interferons, such as Pegasys using permanent branched PEG or Peg-Intron using linear PEG for Interferon-α conjugation only retain 7 and 28% specific antiviral activity of the unmodified Interferon α-2a and Interferon α-2b, respectively. For many therapeutic proteins, a significant loss in biological activity can result in a poor PK-PD profile which often limits the therapeutic application of Pegylation. The numbers and sizes of PEG have significant impacts on proteins' biological activity, pharmacokinetic and pharmacodynamic properties.
Another question that remains unanswered in the art is whether prolonging half-lives of short-lived proteins sequentially in vivo, by allowing controlled degradation of the polymeric linkages would prolong therapeutic drug availability in vivo, thus improving clinical outcome. Given the above, it is desirable to have biomolecule-polymer conjugates that overcome these deficiencies. The instant invention remedies these shortcomings in the prior art.